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A detection method for amplifying internal standard and bacterial multiple PCR

A technology for amplifying internal standards and nucleotide sequences, applied in the field of amplification of internal standards and bacterial multiplex PCR, can solve problems such as false negatives, missed detections, food safety and people's health threats, and reduce the difficulty of synthesis , The overall length is short, the detection effect is stable and the accuracy is high.

Inactive Publication Date: 2019-03-19
山东省畜产品质量检测中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Although the PCR method is fast and sensitive, when there are inhibitors in the amplification system, the results often appear false negative, which means missed detection, which poses a potential threat to food safety and people's health

Method used

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  • A detection method for amplifying internal standard and bacterial multiple PCR
  • A detection method for amplifying internal standard and bacterial multiple PCR
  • A detection method for amplifying internal standard and bacterial multiple PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] 1. Acquisition of internal standard for competitive amplification

[0066] 1. Select the amplified internal standard DNA sequence

[0067] Through DNAman software sequence comparison, take a DNA sequence in the pREST-B plasmid that has no homology with the pMD18-T plasmid, and carry out Blast homology on this fragment with the target bacterial genome (taking Salmonella enteritidis and Escherichia coli as examples) on NCBI In contrast, the homology with the invA gene of Salmonella enteritidis is 3.61%, and the homology with the E. coli O157:H7fliC gene is 4.42%. The primers are designed based on a sequence with very low homology, and the amplified fragment of the primers is non-competitive. Amplify the internal standard DNA sequence.

[0068] 2. pREST-B plasmid culture and extraction

[0069] Bacteria containing the pREST-B plasmid were cultured in LB medium supplemented with ampicillin sodium, and the pREST-B plasmid was extracted using a kit method.

[0070] 3. Prim...

Embodiment 2

[0098] Embodiment 2 Optimum Concentration Determination

[0099] The Salmonella Enteritidis strain and Escherichia coli were respectively diluted 10-fold with sterile water to obtain 10 strains of Salmonella Enteritidis. 3 CFU / ml, Escherichia coli O157:H7 10 3 CFU / ml, Salmonella Enteritidis 10 2 CFU / ml, Escherichia coli O157:H710 2 CFU / ml, Salmonella Enteritidis 10 1 CFU / ml, Escherichia coli O157:H710 1 CFU / ml.

[0100] The internal standard for the competitive amplification of the designed Salmonella enteritidis strain and Escherichia coli was determined to be 40 ng / μl by a nucleic acid protein analyzer, and then diluted 10 times with sterile ultrapure water to obtain a concentration of 4 fg / μl , 0.4fg / μl competitive amplification internal standard.

[0101] The grouping of the optimal concentration experiment is divided into 9 groups, which are Salmonella enteritidis 10 3 CFU / ml, Escherichia coli O157:H710 3 CFU / ml, competitive amplification internal standard mass co...

Embodiment 3

[0119] The influence of embodiment 3 inhibitors on competitive amplification internal standard-PCR

[0120] Take the poultry meat samples that are negative for Salmonella Enteritidis and Escherichia coli O157:H7 after the traditional culture method (national standard inspection method) and PCR detection, suspend in sterilized water (50:50, W / V), homogenize for 2min, 13000r / min centrifugation for 2min, and store the supernatant as the PCR detection inhibitor solution. The inhibitor is diluted with ultrapure water, to 1 / 64, 1 / 32 of the original concentration, and detected according to the prior art and the method of the present invention respectively, to determine its effect on the competitive amplification internal standard-PCR The effect of system amplification.

[0121] The detection method for carrying out multiple PCR on the above samples comprises the following steps:

[0122] The Salmonella Enteritidis strain and Escherichia coli were respectively diluted 10-fold with ...

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Abstract

The invention relates to an amplification interior label and multi-bacterial PCR detection method. The nucleotide sequence of the amplification interior label is as shown in SEQ ID NO.1. The detection method comprises the following steps: (1) extracting genome DNA of a to-be-detected sample, so as to prepare a DNA solution; (2) by taking the genome DNA as a template, adding the amplification interior label into the PCR amplification system for PCR amplification, thereby preparing a PCR product; and (3) judging whether false positive result happens according to the PCR product after agarose gel electrophoresis. Instead of the situation that in the prior art different amplification interior labels are fused for different target bacteria, the amplification interior label is shared by multiple target bacteria in multi-bacterial PCR detection, the synthesis difficulty is greatly reduced as the overall length is small, and moreover the detection effect stability and the accuracy degree are high.

Description

technical field [0001] The invention relates to a detection method for amplifying an internal standard and bacterial multiplex PCR, and belongs to the technical field of gene detection. Background technique [0002] Although the PCR method is fast and sensitive, when there are inhibitors in the amplification system, the results often have false negatives, which means missed detection, which poses a potential threat to food safety and people's health. [0003] The amplified internal standard is a piece of artificially constructed DNA sequence or a piece of conserved gene sequence of pathogenic bacteria added to the PCR reaction system to indicate false negative phenomena. The main principle of the amplification internal standard is: add the amplification internal standard to the PCR reaction system to make it co-amplify with the target gene. If there are some inhibitory factors in the reaction system, the internal standard and the target gene will be amplified. All amplifica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10C12N15/11C12R1/19C12R1/42
CPCC12Q1/686C12Q1/689C12Q2600/166C12Q2537/143C12Q2545/107C12Q2565/125
Inventor 高迎春杨林魏秀丽薄永恒
Owner 山东省畜产品质量检测中心